Comparative genomics of sex chromosomes in Diptera: gene trafficking, dosage compensation, sex-biased expression and gene content evolution. Sex chromosomes are derived from ordinary autosomes but harbor many unique characteristics, including a nonrandom gene content, or sex-specific expression patterns. However, most of our knowledge of sex chromosome biology is restricted to a few model species, including humans or Drosophila, since identification of sex-linked genes traditionally requires laborious segregation analysis or physical mapping. Research in these two groups has revealed that sex chromosomes often show unusual, yet sometimes highly discordant patterns of genome evolution and function. In particular, X chromosomes of mammals show an excess of male genes, yet a deficiency in Drosophila; the X is dosage compensated through X inactivation in female mammals, yet is hyper-transcribed in Drosophila males; the gene content is highly conserved on the Y chromosome in primates and many Y genes have homologs on the X, yet turns over rapidly in Drosophila with no remaining X-Y homology. Thus, to disentangle species-specific patterns from general features of sex chromosomes, it is necessary to compare the genomic properties of several independently formed sex chromosomes. We have developed a new powerful statistical method for identifying a large number of sex-linked genes that is applicable to virtually any organism using next-generation sequencing techniques. This will allow us to examine sex chromosomes in several non-model organisms, and study the functional and evolutionary properties of sex chromosomes in an unprecedented way. We propose to use broad phylogenetic sampling in Diptera and novel sequencing-based methods to identify sex-linked sequences, combined with transcriptome profiling across sexes and tissues, and investigation of the sex-specific chromatin structure. This rich data set will allow us to identify transitions among sex chromosomes and sex determining mechanisms, and will reveal key principals that shape the evolution and function of independently evolved sex chromosomes in flies, including identification of the general mechanisms of dosage compensation and the evolution of sex-biased expression patterns.